Nebulized amiloride has been proposed as therapy in cystic fibrosis to block Na⁺ hyperabsorption in airway epithelium and prevent dehydration of secretions. Patients with cystic fibrosis often have reactive airways. Bovine and canine trachea relax to amiloride in vitro, suggesting another benefit as a bronchodilator, whereas guinea pig trachea, a useful model of human airways, does not. We hypothesized that human airways would respond like guinea pig airways. Airway ring segments from guinea pigs, mice, and human fetuses were constricted with the concentration of acetylcholine producing 50-75% maximum contraction. Subsequent changes in isometric tension to cumulative additions of amiloride (10⁸-10⁴ M) were measured. Guinea pig airways contracted 29 ± 5%, mouse airways contracted 23 ± 6%, and human fetal airways contracted 30 ± 8%. Contraction to amiloride was mimicked by dimethylamiloride, a more selective inhibitor of the Na⁺/H⁺ antiporter, and was attenuated by protein kinase C (PKC) inhibition with GF109203X and staurosporine. The present study indicates that amiloride-induced airway contraction in guinea pigs and mice closely parallels the response in isolated human airways and that the mechanism may involve the Na⁺/H⁺ antiporter and PKC.